The present invention relates to conjugates having prolonged hydrolyzing activity in vivo against glycolipids such as glucocerebroside.
Gaucher's Disease is an autosomal recessive genetic disorder which effects about 20,000 people in the Unites States. The disease is the most common lysosomal storage disorder and describes a defect in the afflicted's naturally-occurring glucocerebrosidase (GC). This defect causes pathological storage of the complex lipid, glucocerebroside, primarily in organs and tissues of the reticuloendothelial system. The disease is systemic and patients may experience enlargement of the liver and spleen as well as replacement of the bone marrow with lipid-filled cells known as Gaucher cells.
Unfortunately, there is currently no cure for patients suffering from this disease. Treatment for the disease is largely symptomatic. For example, analgesics are used for relief of pain, blood and platelet transfusions are often indicated. In cases where the disease is severe, a splenectomy is indicated to remove the enlarged spleen.
Gaucher's Disease is considered to be a good candidate for enzyme-replacement therapy. For example, U.S. Pat. No. 3,910,822 discloses the use of GC isolated from human placental tissue as a treatment of Gaucher's Disease. In addition, PCT Publication Nos. WO 90/07573 and WO 89/05850 describe preparing GC using recombinant DNA techniques. While these advancements are significant, effective treatment and management of the disease for many patients remains elusive. A chief drawback with current therapies is the relatively short period of time that the replacement enzyme is active in vivo. Accumulated glucocerebroside is not always fully metabolized. Thus, the long term effects of the lingering glycoprotein has not been addressed.
U.S. Pat. No. 4,935,465 describes protein conjugates including glucocerebrosidase reversibly linked to water soluble polymers. The linking groups described therein are based on maleic acid derivatives which are quickly hydrolyzed in vivo and thus release the unmodified protein from the polymer. The present inventors, however, have found that alternative linking groups having a much greater resistance to in vivo hydrolysis provide conjugates which are enzymatically-active over longer periods and thus are better suited to act on accumulated glycolipids and thus reduce the residual amounts of glucocerebroside in the plasma and normalize spleen, liver and skeletal abnormalities.
In spite of the recombinant enzyme's apparent homology with human enzymes, one shortcoming is the fact that recombinantly prepared GC has been found to have low levels of activity at lysosomal pH levels which tend to be from about 4.0 to 5.0. For example, it has been determined that some recombinantly prepared GC have activity levels of only 10% at pH 4.0 and 30% at pH 4.5 (where activity at the optimal pH of about 5.5 is defined as 100%). In view of the fact that Gaucher's Disease is a lysosomal storage disorder, improving the activity of a recombinant enzyme at lower pH's would be a significant advance. Yet another shortcoming associated with recombinantly prepared GC is its suboptimal activity at pH ranges associated with arterial and capillary regions, typically about 7.4.+-.0.5. Any increase in enzyme activity of exogenous enzyme replacements would enhance treatment.